In our earlier studies we have developed experimental models for the triggering of symptoms in both premenstrual dysphoric disorder (PMD) and postpartum depression that we continue to employ in our efforts to identify both the underlying biology of these conditions as well as the development of new safe and effective therapies for women with these conditions. We have extended and replicated our earlier findings by demonstrating that women with PMD (n = 35), who respond to gonadotropin releasing hormone (GnRH) agonist-induced ovarian suppression experience a recurrence of PMD after the initial re-exposure to combined estradiol and progesterone. However, recurrent symptoms do not occur once hormone levels are stabilized over the subsequent two months of continuous replacement therapy. These observations are of both clinical and scientific importance, as they identify promising phenotypes and suggest the physiologic basis for the susceptibility to experience PMD and will also provide alternative hormone-based therapies for women with this condition. In addition to our studies on the behavioral effects of changes in ovarian steroids across the menstrual cycle and during the postpartum, we employ methodologies to investigate the underlying biological mechanisms of these conditions including studies investigating HPA axis responsivity and those employing positron emission tomography (PET), structural magnetic resonance imaging (MRI), and functional magnetic resonance imaging (fMRI). First, we hypothesized that women with PMDD would show abnormalities of HPA axis function analogous to those reported in depressive illness, and that ovarian steroids would differentially regulate HPA axis function in women with PMDD compared with asymptomatic controls. Eighteen women with PMDD and twenty-five controls underwent an open-label trial of the GnRH agonist depot Lupron with ovarian steroid replacement administered in a double-blind crossover design. HPA axis function was similar in PMDD compared with controls. In all, progesterone significantly increased the secretion of cortisol and ACTH compared with estradiol and hypogonadism. Our data demonstrate that HPA axis regulation is normal in PMD, suggesting that the pathophysiology of PMD differs from major depression. Second, our neuroimaging studies have identified both a neural substrate of risk in PMDD as well as a brain-region specific response that could underlie the differential behavioral response to ovarian steroids in this condition. We performed O15 PET and fMRI studies in women who are participating in the GnRH agonist-induced hypogonadism study. In this study, cognitive activation was achieved using the N-back test which allows us to vary the cognitive load and the effort required to perform the task. We observed that women with PMD show abnormal prefrontal recruitment, specifically greater activation than controls throughout the DLPFC bilaterally. These findings are robust and observable in both 015 PET and fMRI techniques. In both imaging modalities, significantly increased DLPFC activations in PMDD compared with controls occurred in all three hormone conditions and, therefore, were independent of hormone condition. Moreover, DLPFC activations in PMDD correlated negatively with measures of PMD functional impairment (i.e., global assessment of functional impairment GAF scores), age of onset of PMDD, and baseline (prior to Lupron) symptom severity measures - the greater the over-activation, the greater the disability, the earlier age of onset, and the more severe baseline symptomatology) Our findings suggest that these persistent effects, even in the clinically asymptomatic phase of the hormone-manipulation paradigm(i.e., Lupron alone), may represent an enduring, trait-like predisposition to this hormonally-triggered disorder. By identifying the prefrontal cortex, specifically the DLPFC, as key in the pathophysiology of PMDD, these data provide insight into potential neural mechanisms contributing to this disorder. Additionally, in studies of resting state activations, we observed a significant interaction between diagnosis and hormone condition in several affect-related regions, including subgenual cingulate, medial orbitofrontal cortex, and hippocampus. Women with PMDD had greater resting rCBF during Lupron alone (i.e., the asymptomatic state in PMDD) in the aforementioned brain regions compared with both progesterone and estradiol replacement (when symptoms can recur in PMD); whereas controls showed the opposite pattern. These data demonstrate that in PMD, ovarian hormones differentially impact resting rCBF in regions typically involved in the pathophysiology of affective disorders. These data provide a framework for understanding control of affective state and propensity for mood destabilization in this condition. Future findings in these studies may identify disturbances of a process that is critical to the clinical phenomenology of PMD, a functional disturbance of the normal modulatory effect of gonadal steroids, and a locus of the disturbance. We collaborate with Drs. Rima Kaddurah-Daouk at Duke University and Thomas Hankemeier at Leiden University to more comprehensively examine the profile of steroid and steroid metabolites with a metabolomics platform employing liquid chromatography-tandem mass spectroscopy. Our strategy is to perform metabolomic studies in women with PMD who respond to Lupron with elimination of symptoms and who experience return of symptoms during progesterone or estradiol replacement. Thus, we wish to specifically examine the effects of both estradiol and progesterone on the pattern of steroid metabolites in women with PMD and an asymptomatic comparison group. Our metabolomic and neuroimaging studies will be complemented by a new series of functional genomic studies performed in collaboration with David Goldmans laboratory at NIAAA, in which we employ lymphoid and fibroblast cell-lines obtained from women with PMD and controls who participate in our Lupron studies. We hypothesize that the capacity for phenotypic differences between women with and those without PMD will be preserved in their cellular function. These experiments allow us to explore the nature of the differential behavioral response by examining protein expression and changes in cellular behaviors associated with the exposure to physiologic levels of either estradiol or progesterone across the two different behavioral phenotypes (women with PMD who experience a remission of PMDD during ovarian suppression and a recurrence of PMDD when ovarian steroids are added back, and controls who are asymptomatic during all three hormone conditions). Preliminary results show that women with PMD express more estrogen receptor (ER) alpha (but not beta) compared with controls (consistent with our findings of differential ER alpha allele frequency in PMD compared with controls observed in a prior study). Additionally, RNA sequencing strategies investigate the response to steroid hormone exposure in immortalized lymphoid cells in these same groups of women. . We observed statistically significant, quantitative and widespread differences in the PMDD transcriptome compared to a carefully selected asymptomatic control group. Our analyses identified several molecular pathways that appear to be altered in women with PMDD; one of these pathways, the chromatin modifying ESC/E(z) complex, is of particular interest as a potential candidate since it is regulated by estradiol and leads to genomic regions of transcriptional repression. The mechanism by which these pathways converge and possibly alter CNS function sufficient to manifest in PMDD will be the focus of our future studies in induced neuronal and glial cells.